Separation of micromolecules

ABSTRACT

This invention is directed to an apparatus and method for the separation of molecules, particularly micromolecules having a molecular mass of less than 5000 Dalton. The present invention is directed to an apparatus for separating micromolecules by electrophoretic separation, the apparatus comprising:  
     (a) an anode;  
     (b) a cathode disposed relative to the anode so as to be adapted to generate an electric field in an electric field area therebetween upon application of a voltage potential between the anode and the cathode;  
     (c) a separation membrane disposed in the electric field area;  
     (d) a first restriction membrane disposed between the anode and the separation membrane so as to define a first interstitial volume therebetween;  
     (e) a second restriction membrane disposed between the cathode and the separation membrane so as to define a second interstitial volume therebetween; and  
     (f) means adapted to provide a sample constituent in a selected one of the first and second interstitial volumes;  
     wherein upon application of the voltage potential, a selected separation product is removed from the sample constituent, thorough the separation membrane, and provided to the other of the first and second interstitial volumes, wherein a micromolecule is capable of being retained in at least one of the interstitial volumes, and wherein a micromolecule is capable of being retained in at least one of the interstitial volumes.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to apparatus and methods for theseparation of molecules, particularly micromolecules having a molecularmass of less than about 5000 Dalton.

[0002] There are increasing numbers of micromolecules being used as foodand diet supplements, pharmaceuticals and neutraceuticals. Increasingnumbers of vitamins, co-factors, plant and microbial extracts are alsobeing developed and used for human and animal consumption. As many ofthese compounds are micromolecules,(having a molecular mass of less thanabout 5000 Dalton (Da)), there is a need to develop methods to separateor purify these compounds in a fast and economical manner. Traditionalseparation methods for micromolecules can alter or denature thesecompounds. Separation methods for larger molecules typically are notconsidered suitable for use in micromolecule separation. Furthermore,traditional methods can be quite time consuming, expensive and difficultto scale up commercially.

[0003] In the past, a preparative electrophoresis technology formacromolecule separation which utilises tangential flow across apolyacrylamide membrane when a charge is applied across the membrane wasused to separate micromolecules. The general design of the earliersystem facilitated the purification of proteins and other macromoleculesunder near native conditions. The technology is bundled into a cartridgecomprising several membranes housed in a system of specially engineeredgrids and gaskets which allow separation of macromolecules by chargeand/or molecular weight. The system can also concentrate anddesalt/dialyse at the same time. The multi-modal nature of the systemallows this technology to be used in a number of other areas especiallyin the production of biological components for medical use. Thetechnology isolates macromolecules using the duality of charge and size.However, the technology could not be extended to the isolation ofmolecules below about 5000 Da. This meant that while molecules smallerthan 5000 Da could be removed using at least charge-based separation,the resulting target molecule could not be captured.

[0004] The separation of micromolecules, molecules deemed to be lessthan about 5 kDa, was previously thought not to be possible usingelectrophoresis technology devised to separate macromolecules. This wasdue to the limit in pore size of membranes normally used in the systems.For example, the smallest cut-off produced in polyaccrylamide membranesis about 5 kDa which will retain any molecule larger than 5 kDa.

[0005] There were several problems encountered in the separation ofmicromolecules using an unmodified electrophoresis system. Difficultyretaining micromolecules in the system has been overcome with theaddition of combinations of membranes. However, these membranesthemselves posed problems in that they are not designed to retainliquids and can produce large levels of electro-endo-osmosis. The liquidretention problem has been solved by backing the membranes with thehydrogel polyacrylamide membranes, which also helped to reduce theelectro-endo-osmosis levels.

[0006] It is desirable to have a preparative electrophoresis systemwhich can efficiently and effectively remove micromolecules.

[0007] The subject invention overcomes the above limitations and others,and teaches an electrophoresis system, which can be scaled up forpreparative applications, which apparatus can efficiently andeffectively separate micromolecules.

SUMMARY OF THE INVENTION

[0008] In accordance with the present invention, there is provided anelectrophoresis system which efficiently and effectively separatemicromolecules.

[0009] Further, in accordance with the present invention, there isprovided an apparatus for separating micromolecules by electrophoreticseparation, the apparatus comprising:

[0010] (a) an anode;

[0011] (b) a cathode disposed relative to the anode so as to be adaptedto generate an electric field in an electric field area therebetweenupon application of a voltage potential between the anode and thecathode;

[0012] (c) a separation membrane disposed in the electric field area;

[0013] (d) a first restriction membrane disposed between the anode andthe separation membrane so as to define a first interstitial volumetherebetween;

[0014] (e) a second restriction membrane disposed between the cathodeand the separation membrane so as to define a second interstitial volumetherebetween; and

[0015] (f) means adapted to provide a sample constituent in a selectedone of the first and second interstitial volumes;

[0016] wherein upon application of the voltage potential, a selectedseparation product is removed from the sample constituent, thorough theseparation membrane, and provided to the other of the first and secondinterstitial volume and wherein a micromolecule is capable of beingretained in at least one of the interstitial volumes.

[0017] Still further, in accordance with the present invention, there isprovided an apparatus for separating micromolecules by electrophoresis,the apparatus comprising:

[0018] (a) an anode buffer compartment and a cathode buffer compartment;

[0019] (b) electrodes positioned in the buffer compartments;

[0020] (c) a first chamber and a second chamber positioned on eitherside of an ion-permeable separation membrane having a defined molecularmass cut-off, the first chamber and the second chamber being positionedbetween the anode and the cathode buffer compartments and separated byan ion-permeable restriction membrane positioned on each side of theseparation membrane, the restriction membrane(s) allowing flow of ionsinto and out of the compartments and chambers under the influence of anelectric field but substantially restrict movement of at least onemicromolecule type from the second chamber into the buffer compartment.

[0021] Preferably, the buffer compartments, the first chamber and thesecond chamber are configured to allow flow of the respective buffer,first and second solutions forming streams. In this form, large volumescan be processed quickly and efficiently. The solutions are typicallymoved or recirculated through the compartments and chambers fromrespective reservoirs by pumping means. Peristaltic pumps have beenfound to be particularly suitable for moving the fluids.

[0022] Preferably, the ion-permeable separation membrane has a molecularmass cut-off greater than the molecular mass of the micromolecule to beseparated.

[0023] An advantage of the present invention is that micromolecules canbe separated efficiently and effectively using preparativeelectrophoresis under near native conditions which results in higheryields and excellent recovery.

[0024] Another advantage of the present invention is that the system issuitably used in a number of other areas, especially in the productionof biological components for medical use.

[0025] Another advantage of the present invention is that the system canbe suitably configured to remove biological contaminants at the point ofseparation.

[0026] These and other advantages and benefits of the invention will beapparent to those skilled in the art upon reading and understanding ofthe following detailed description.

[0027] BRIEF DESCRIPTION OF DRAWINGS

[0028]FIG. 1 shows the transfer of BB-FCF through the 5 kDa separationmembrane from the sample stream (US) where it transiently builds up inthe product stream (DS). After 20 minutes all the BB-FCF had transferredthrough the bottom restriction membrane into the buffer stream where itwas lost.

[0029]FIG. 2 shows Azorubine transfer using only polyacrylamidemembranes in the Gradiflow system. Transfer from the sample (US) to theproduct stream (DS) occurred but the molecules only built up for a smallperiod of time before moving completely into the buffer stream.

[0030]FIG. 3 shows that Biotin behaves in a similar manner to the othertested micromolecules in that it transferred rapidly from the samplestream (US) and appeared transiently in the product stream (DS) beforeeluting into the buffer stream.

[0031]FIG. 4 shoes that phytoestrogen transferred into the productstream (DS) from the sample stream (US), where it built up and over timebefore dissipating into the buffer stream.

[0032]FIG. 5 shows BB-FCF separated with 74% yield in one hour. Themolecules were readily captured using the 1 kDa ultrafiltration membranein the cartridge.

[0033]FIG. 6 shows the level of Azorubine in the sample stream (US)decreased over time and transferred to the product stream (DS). A totalof 83% of the Azorubine was transferred and retained in 45 minutes.

[0034]FIG. 7 shows Biotin was readily transferred from the sample streaminto the product stream and collected with high yield (84%) in theproduct stream.

[0035]FIG. 8 shows phytoestrogen was transferred from the sample streaminto the product stream where it could be collected.

[0036]FIG. 9 shows separation of BB-FCF from Azorubine in a systemadapted to carry out the method according to the present invention. TheBB-FCF was retained in the sample stream whilst the Azorubine was movedto the product stream.

[0037]FIG. 10 shows BB-FCF was separated from Azorubine using a sizeexclusion approach with retention of 74% of the BB-FCF. Only a smallpercentage of the Azorubine remained with the BB-FCF after three hoursof separation.

[0038]FIG. 11 is a schematic view of a preferred embodiment of theseparation apparatus of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0039] This invention is directed to an apparatus and method for theseparation of molecules, particularly micromolecules having a molecularmass of less than about 5000 Dalton. The present invention is directedto an apparatus for separating micromolecules by electrophoreticseparation, the apparatus comprising:

[0040] (a) an anode;

[0041] (b) a cathode disposed relative to the anode so as to be adaptedto generate an electric field in an electric field area therebetweenupon application of a voltage potential between the anode and thecathode;

[0042] (c) a separation membrane disposed in the electric field area;

[0043] (d) a first restriction membrane disposed between the anode andthe separation membrane so as to define a first interstitial volumetherebetween;

[0044] (e) a second restriction membrane disposed between the cathodeand the separation membrane so as to define a second interstitial volumetherebetween; and

[0045] (f) means adapted to provide a sample constituent in a selectedone of the first and second interstitial volumes;

[0046] wherein upon application of the voltage potential, a selectedseparation product is removed from the sample constituent, thorough theseparation membrane, and provided to the other of the first and secondinterstitial volumes, and wherein a micromolecule is capable of beingretained in at least one of the interstitial volumes.

[0047] In a preferred embodiment, the present invention is directed anapparatus for separating micromolecules by electrophoresis, theapparatus comprising:

[0048] (a) an anode buffer compartment and a cathode buffer compartment;

[0049] (b) electrodes positioned in the buffer compartments;

[0050] (c) a first chamber and a second chamber positioned on eitherside of an ion-permeable separation membrane having a defined molecularmass cut-off, the first chamber and the second chamber being positionedbetween the anode and the cathode buffer compartments and separated byan ion-permeable restriction membrane positioned on each side of theseparation membrane, the restriction membrane(s) allowing flow of ionsinto and out of the compartments and chambers under the influence of anelectric field but substantially restrict movement of at least onemicromolecule type from the second chamber into the buffer compartment.

[0051] Preferably, the buffer compartments, the first chamber and thesecond chamber are configured to allow flow of the respective buffer,first and second solutions forming streams. In this form, large volumescan be processed quickly and efficiently. The solutions are typicallymoved or recirculated through the compartments and chambers fromrespective reservoirs by pumping means. Peristaltic pumps have beenfound to be particularly suitable for moving the fluids.

[0052] Preferably, the ion-permeable separation membrane has a molecularmass cut-off greater than the molecular mass of the micromolecule to beseparated.

[0053]FIG. 11 shows a preferred embodiment of the apparatus 10 of thepresent invention. The apparatus 10 includes an anode buffer zone orcompartment 11 and a cathode buffer zone or compartment 12 separated byan ion-permeable separation barrier 13. Electrodes 14 and 15 areprovided inside the buffer zones or compartments so as to be on oppositesides of the separation membrane 13. It is understood, however, that inanother embodiment, the electrodes are positioned outside the buffercompartments. The electrodes are used to apply an electrophoreticpotential across the separation membrane.

[0054] A first chamber 16 is positioned between the anode buffercompartment 11 and the separation membrane 13. The first chamber isdefined on one side by the separation membrane 13 and on the other sideby a first restriction membrane 18. It is understood, however, that inanother embodiment, the first chamber is positioned between the cathodebuffer compartment and the separation membrane. In one embodiment, thefirst restriction membrane is comprised of at least two membranes 18 and18 b having distinctive pore sizes.

[0055] A second chamber 17 is positioned between the cathode buffercompartment 12 and the separation barrier 13. The second chamber isdefined on one side by the separation membrane 13 and on the other sideby a second restriction membrane 19 on the other side. It is understood,however, that in another embodiment, the second chamber is positionedbetween the anode buffer compartment and the separation membrane. In oneembodiment, the second restriction membrane is comprised of at least twomembranes 19 a and 19 b having distinctive pore sizes.

[0056] The apparatus is further comprised of switch 25 for selection ofthe application of a voltage source (such as to turn the voltage sourceoff or have resting periods), switch 26 to switch current direction forcathode/anode or to have reversal periods, and voltage sources 27 and28.

[0057] The anode buffer compartment and the cathode buffer compartmentare supplied with suitable buffer solutions by any suitable means. Amixture comprising micromolecules is supplied directly to the firstchamber by any suitable means. The micromolecules are separated from thesecond chamber by any suitable means.

[0058] Preferably, the buffer compartments, the first chamber and thesecond chamber are configured to allow flow of the respective buffer,sample and product solutions forming streams. In this form, largevolumes can be processed quickly and efficiently. The solutions aretypically moved or recirculated through the compartments and chambersfrom respective reservoirs by pumping means. In a preferred embodiment,peristaltic pumps are used as the pumping means for moving the fluids.

[0059] The buffer, sample or product solutions are cooled by anysuitable means to ensure no inactivation of the micromolecules occursduring the separation process and to maintained a desired temperature ofthe apparatus while in use.

[0060] Preferably, in order to collect and concentrate the separatedmicromolecules, solution in the product chamber or stream is collectedand replaced with suitable solvent to ensure that electrophoresis cancontinue.

[0061] Preferably, at least one restriction membrane is formed as acomposite or sandwich arrangement with at least two materials.Preferably, at least one restriction membrane is formed as a sandwicharrangement with at least two layers of material. In this preferredform, the sandwich arrangement includes an inner layer (facing theseparation membrane in the first and second solvent streams,respectively) comprising a membrane having a pore size with a molecularmass cut-off less than the about 5000 Da and an outer layer comprising amembrane having a molecular mass cut-off of greater than about 5000 Da.

[0062] In a preferred form, the inner layer is made from anultrafiltration, electrodialysis or haemodialysis material and the outerlayer is made from polyacrylamide. In this preferred arrangement, theouter layer provides some structural support for the filtration membranewhile preventing unwanted movement of fluid. The pore size of thefiltration membrane is selected according to the size of themicromolecule to be separated such that the micromolecule cannot passthrough the membrane. Typically, the molecular mass cut-off of thefiltration membrane is between about 100 Da to 5000 Da. More preferably,the molecular mass cut-off is around 200 Da.

[0063] Hydrogel ion-permeable separation membranes (an ultrafiltration,electrodialysis and/or haemodialysis membranes coated withpolyacrylamide) would be an alternative membrane type suitable for thepresent invention. Such membranes are possible to manufacture, but arecurrently not commercially available.

[0064] Preferably, the ion-permeable separation barrier is a membranemade from polyacrylamide and having a molecular mass cut-off from about5 to 1000 kDa. The size of the separation membrane cut-off will dependon the sample being processed and the other molecules in the mixture.

[0065] The restriction barriers or membranes positioned adjacent thesample and product chambers can have the same molecular mass cut-off ordifferent cut-offs therefore forming an asymmetrical arrangement.Typically, the restriction membrane separating the product chamber fromthe buffer compartment is formed in a sandwich configuration.

[0066] The distance between the electrodes can have an effect on theseparation or movement of micromolecules through the barriers. It hasbeen found that the shorter the distance between the electrodes, thefaster the electrophoretic movement of micromolecules. A distance ofabout 6 cm has been found to be suitable for a laboratory scaleapparatus. For scale up versions, the distance will depend on the numberand type of separation membranes, the size and volume of the chambersfor samples, buffers and separated products. Preferred distances wouldbe in the order of 6 cm to about 10 cm. The distance will also relate tothe voltage applied to the apparatus. The effect of the electric fieldis based on the equation:

e=V/d

[0067] (e=electric field, V=voltage, d=distance)

[0068] Therefore, smaller distances between the electrodes arepreferred. Preferably, the distance between the electrodes shoulddecrease in order to increase electric field strength, thereby furtherimproving transfer rates.

[0069] Flow rate of sample/buffer can have an influence on theseparation of micromolecules. Rates of milliliters per hour up to litersper hour can be used depending on the configuration of the apparatus andthe sample to be separated. Currently in a laboratory scale instrument,the preferred flow rate is about 20±5 mL/min. However, flow ratesranging from about 0 to about 50,000 mL/min are also used across thevarious separation regimes. In some embodiments the maximum flow rate ishigher, depending on the pumping means and size of the apparatus. Theflow rate is dependent on the product to be transferred, efficiency oftransfer, pre- and post-positioning with other applications.

[0070] Voltage and/or current applied can vary depending on theseparation. Typically up to several thousand volts may be used butchoice and variation of voltage will depend on the configuration of theapparatus, buffers and the sample to be separated. In a laboratory scaleinstrument, the preferred voltage is about 250 V. However, depending ontransfer, efficiency, scale-up and particular method about 0 to about5000 are used. Higher voltages may also be considered, depending on theapparatus and sample to be treated.

[0071] A number of first and second chambers could be stacked in the oneapparatus for use in a scale-up device.

[0072] A single stream configuration can be produced where the secondchamber forms a buffer chamber. In this configuration, contaminantswould be moved out of the first chamber into the buffer compartments andthe product of interest retained in the first chamber. In single streamconfiguration, the membranes can have either a symmetric or asymmetricarrangements. The present invention also includes these embodiments.

[0073] In use, a sample containing one or more micromolecules is addedto the first chamber and an electric potential is applied to causemovement of at least one micromolecule from the sample through theseparation membrane into the second chamber while the restrictionmembranes prevent movement of micromolecules from the first or thesecond chambers into the respective electrophoresis buffer chambers.Preferably, the micromolecule is removed and collected from the productchamber.

[0074] In a second aspect, the present invention provides a separationcartridge suitable for use in an electrophoresis apparatus forseparating micromolecules, the cartridge comprising:

[0075] (a) a housing;

[0076] (b) an ion-permeable separation membrane having a definedmolecular mass cut-off positioned in the housing;

[0077] (c) an ion-permeable restriction membrane positioned on eitherside of the separation membrane in the housing and spaced to form afirst chamber and second chamber on either side of the separationmembrane, wherein the restriction membrane is adapted to allow flow ofions into and out of the compartments and chambers under the influenceof an electric field but substantially restrict movement of at least onemicromolecule type from the second chamber. In a preferred embodiment,the cartridge further includes:

[0078] (d) electrodes positioned in the housing on the outer sides ofthe restriction barriers.

[0079] Preferably, the separation barrier is a membrane composed ofpolyacrylamide and having a molecular mass cut-off from about 5 to 1000kDa.

[0080] Preferably, the ion-permeable separation membrane has a molecularmass cut-off greater than the molecular mass of the micromolecule to beseparated.

[0081] At least one restriction membrane is preferably formed as asandwich or composite arrangement of membranes with at least twomaterials. Preferably, the sandwich arrangement includes an inner layercomprising a restriction membrane having a pore size with a molecularmass cut-off less than about 5000 Da and an outer layer comprising arestriction membrane having a molecular mass cut-off of greater thanabout 5000 Da.

[0082] In a preferred form, the inner layer is made from anultrafiltration, electrodialysis or haemodialysis material and the outerlayer is made from polyacrylamide. In this preferred arrangement, theouter layer provides some structural support for the filtration membranewhile preventing unwanted movement of fluid. The pore size of thefiltration membrane is selected according to the size of themicromolecule to be separated such that the micromolecule cannot passthrough the membrane. Typically, the molecular mass cut-off of thefiltration membrane is between about 100 Da to 5000 Da. More preferably,the molecular mass cut-off is around 200 Da.

[0083] In a third aspect, the present invention provides a method ofseparating a micromolecule from a liquid sample, the method comprising:

[0084] (a) providing an electrophoresis apparatus according to the firstaspect of the present invention;

[0085] (b) placing the sample in the first chamber of the apparatus;selecting a solvent for the first chamber having a pH such that themicromolecule to be separated is charged;

[0086] (c) applying an electric potential between the first and secondchambers causing movement of micromolecules in the first chamber throughthe separation membrane into the second chamber while unwanted moleculesare substantially prevented from entering the second chamber;

[0087] (d) optionally, periodically stopping and reversing the electricpotential to cause movement of molecules having entered the separationmembrane to move back into the first chamber, while substantially notcausing any micromolecules that have entered the second chamber tore-enter first chamber; and

[0088] (e) maintaining steps (c) and optionally (d) until the desiredamount of micromolecules are moved to the second chamber.

[0089] The micromolecule can be any micromolecule capable of receivingor having a charge. Examples include, but not limited to, biotin,Brilliant Blue FCF (BB FCF), azorubine, phytoestrogen, digoxigenin,hormones, cytokines, dyes, vitamins, chemicals, neutraceuticals,pharmaceuticals along with food and diet supplements.

[0090] The sample can contain one or more micromolecules of interest.Examples include, but are not limited to, crude extracts, microbialcultures, cell lysates, cellular products, chemical processing mixtures,cell culture media, plant products or extracts.

[0091] Solvent in the form of buffers that have been found to beparticularly suitable for the method according to the present inventionare Tris Borate around pH 9. It will be appreciated, however, that otherbuffers or solvents would also be suitable, depending on the separation.The concentration of the selected buffers can also influence or effectthe movement of micromolecules through the separation barrier. Typicallyconcentrations of about 10 mM to about 200 mM, more preferably 20 mM to80 mM, have been found to be particularly suitable. Almost any buffersand/or solvents can be used with the present invention. The buffersand/or solvents that can be used are procedure/method/separationdependent. The concentration of the buffer and/or solvent is dependentupon the application/separation/procedure.

[0092] Reversal of current is an option but another embodiment is aresting period. Resting (a period without an electric potential beingapplied, but pumps remain on) is an optional step that can replace or beincluded before or after an optional electrical potential reversal. Thisreversal technique is often practised for protein separation work as analternative to reversing the potential.

[0093] One benefit of the method according to the present invention isthe possibility of scale-up without denaturing or adversely altering thephysical or biological properties of the micromolecule.

[0094] In a fourth aspect, the present invention provides amicromolecule purified or separated by the method according to the thirdaspect of the present invention.

[0095] Preferably the micromolecule is less than 5000 Da. Examplesinclude, but are not limited to, biotin, Brilliant Blue FCF (BB FCF),azorubine, phytoestrogen, digoxigenin, hormones, cytokines, dyes,vitamins, chemicals, neutraceuticals, pharmaceuticals along with fooddiet supplements, and combinations thereof.

[0096] In a fifth aspect, the present invention relates to use of themicromolecule according to the fourth aspect of the present invention indietary, medical and veterinary applications.

[0097] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement, integer or step, or group of elements, integers or steps, butnot the exclusion of any other element, integer or step, or group ofelements, integers or steps. Any discussion of documents, acts,materials, devices, articles or the like which has been included in thepresent specification is solely for the purpose of providing a contextfor the present invention. It is not to be taken as an admission thatany or all of these matters form part of the prior art base or werecommon general knowledge in the field relevant to the present inventionas it existed in Australia before the priority date of each claim ofthis application.

[0098] In order that the present invention may be more clearlyunderstood, preferred forms will be described with reference to thefollowing drawings and examples.

MODES FOR CARRYING OUT THE INVENTION

[0099] The separation of micromolecules, molecules deemed to be lessthan about 5 kDa, was previously thought not to be possible usingelectrophoresis technology devised to separate macromolecules. This wasdue to the limit in pore size of membranes normally used in the systems.For example, the smallest cut-off produced in polyaccrylamide membranesis about 5 kDa which will retain any molecule larger than 5 kDa. Thepresent invention results from modification of earlier technology to becapable of separating micromolecules by using some barriers or membranesother than polyacrylamide membranes traditionally used. It has beenfound that when commercially available membranes in the form ofultrafiltration, electrodialysis and haemodialysis membranes are used asseparation or restriction membranes, the size of molecule that can bedealt with, is significantly smaller than previously thought.

[0100] In order to separate and purify micromolecules, hydrogelpolyacrylamide membranes traditionally used in the earlier system'scartridge were placed as backing to a commercial membrane with thedesired pore size. The polyacrylamide membrane is useful to preventunregulated fluid movement across the membranes, whilst the commercialmembrane is used to retain the smaller molecular species within thesample or product streams or chambers. The membranes used for this workwere Pall Gelman Omega ultrafiltration membranes. These ultrafiltrationmembranes are available commercially with pore sizes ranging fromhundreds of kDa down to 1000 Da.

[0101] Another surprising finding was that the relative pore size of theultrafiltration membranes was found to be different when used in thesandwich arrangement than when used in an ultrafiltration unit. Therelative pore size appears to be smaller than the stated size. Thus ithas been shown to be possible to retain molecules as small as 200 Daltonwithin the modified system.

[0102] When an apparatus is operated with the traditional polyacrylamidemembranes used for macromolecule purification or separation, smallmolecules under 5 kDa tend to transfer from the sample stream into theproduct stream where they remain transiently. These molecules then movethrough the bottom restriction membrane into the buffer stream wherethey are lost.

[0103] Experiments were carried out on several model molecules whichhave varied structure and function. The use of these micromoleculesdemonstrates that a wide variety of molecules could be used within thesystem. Two molecules studied wereBis[4-(N-ethyl-N-3-sulfophenylmethyl)aminophenyl]-2-sulfophenylmethyliumdisodium salt (Brilliant Blue FCF) which has a molecular mass of 793Dalton, and Disodium 2-(4-Sulfo-1Napthylazo)-1-Napthol-4-Sulfonate(Azorubine) which has a molecular mass of 502 Dalton. Two othermolecules were also investigated, one being Vitamin H (Biotin) and theother a small phytoestrogen separated from red leaf clover (supplied byNovogen, Sydney Australia). Brilliant Blue FCF and Azorubine are twochemicals currently used in the food industry as colouring agents.Biotin has several uses, first as a necessary vitamin in the human dietand secondly, but not insignificantly, as a labelling agent inscientific assays. Phytoestrogens are separated commercially from manysources primarily soy and clover where they are made into herbal andpharmaceutical medicines.

[0104] Experiments using Brilliant Blue FCF (BB-FCF), Azorubine, Biotinand a Phytoestrogen all confirmed that standard polyacrylamideelectrophoresis membranes do not retain these micromolecules during aseparation. These molecules are very small and definitely consideredmicromolecules; BB-FCF (793 Da), Azorubine (502 Da), Biotin (244 Da) anda Phytoestrogen (˜200 Da).

[0105] Experiments using BB-FCF and Azorubine with a pH 9.0 buffer and acartridge sandwich of 5-5-5 kDa polyacrylamide membranes (upperrestriction 5 kDa, separation membrane 5 kDa, lower restriction 5 kDa)showed that complete transfer from the sample stream occurs in a shortperiod of time. The macromolecules build slightly in the product streambefore passing completely into the buffer stream where they are dilutedso highly that they are lost to analysis. This is also true for thePhytoestrogen and for Biotin.

[0106]FIGS. 1, 2, 3, and 4 show the transient build up and eventual lossof BB-FCF, Azorubine, Biotin, and the Phytoestrogen, within the anapparatus used for macromolecule separation having only polyacrylamidemembranes. The time taken for the micromolecules to completely transferout of the system varies for each molecule. It is likely that this isdue to the difference in charge to mass ratios between the molecules.

[0107] Use of traditional polyacrylamide membranes was therefore foundnot a feasible method for the separation of molecules under about 5 kDa.This led to the novel step of trialing new membranes, previously notconsidered useful for prior art systems. These commercially availablemembranes are manufactured to be used under conditions of high pressurewhere they act purely as a filter. This means that the membranes are notalways “water tight” when used in the earlier system. Only thosemembranes with a very small pore size retain liquid under the lowpressures used in the earlier system. These ultrafiltration membranesare ideal for use in a preparative electrophoresis system unless theyare backed with a membrane designed to stop or reduce transfer of liquidbut allow ion and charged molecule transfer under an electric filed.

[0108] It was found that alternative membrane types could be used whenthey were backed with a hydrogel membrane to give fluid retention. Thisdouble layering not only prevented fluid leakage across the membranesbut also significantly reduced the levels of endo-osmosis produced.Endo-osmosis levels are a big consideration in the design and use ofmembranes for preparative electrophoresis systems. Particularchemistries of membranes can produce such large changes in fluid volumesfrom one stream to another via electro-endo-osmosis so cannot be used.

[0109] The separation and retention of very small molecules was madepossible in the apparatus according to the present invention by using asmall pore sized ultrafiltration membrane (Pall Gelman Omega) as thebottom restriction in combination with a polyacrylamide membrane. Theuse of a 1 kDa Omega ultrafiltration membrane in this position allowedthe capture of molecules as small as 200 Da. Experiments were carriedout again using BB-FCF, Azorubine, Biotin and a Phytoestrogen.

[0110] The following cartridge configuration was used for theseparations:

[0111] 5 kDa polyacrylamide restriction membrane

[0112] Support Grid

[0113] 10 kDa polyacrylamide separation membrane

[0114] Support Grid

[0115] 1 kDa ultrafiltration restriction membrane

[0116] 5 kDa polyacrylamide restriction membrane

[0117] It was possible to move BB-FCF, Azorubine and Biotin using theabove cartridge configuration from the sample stream to the productstream where they were trapped and collected. The Phytoestrogenexperiments used a 5 kDa ultrafiltration membrane to retain themolecules in the product stream.

[0118] Brilliant Blue FCF was readily transferred and retained in theproduct stream with the use of the 1 kDa Omega ultrafiltration membrane.This molecule is only 793 Da and so retention with a 1000 Da membranewould not be expected. The BB-FCF separation is represented by FIG. 5.Analysis of the BB-FCF separation was carried out using the absorbanceat 630 nm.

[0119] Azorubine was moved across the separation membrane and collectedin the product stream. Using a pH 9.0 Tris-Borate buffer 83% of theAzorubine was transferred from the sample stream to the product streamwithin 45 minutes. This transfer was measured using the absorbance ofAzorubine at 516 nm. The separation is illustrated in FIG. 4.

[0120] The Biotin separation utilised a pH 9.0 buffer with the samecartridge configuration as that used for the BB-FCF and Azorubine. Thetransfer of this molecule was monitored using the absorbance at 230 nm.This experiment showed that the 1 kDa ultrafiltration membrane usedcould retain molecules as small as 244 Dalton. FIG. 7 shows that over80% of the Biotin was transferred to the product stream where it wascontained. The phytoestrogen transfer experiment depicted in FIG. 8showed the movement and successful capture of phytoestrogen. Thedecrease over time after the initial high levels of phytoestrogen aremost likely due to the fact that a 5 kDa ultrafiltration membrane wasused as the bottom restriction membrane. The use of a 1 kDaultrafiltration membrane would help to completely retain the smallphytoestrogen.

[0121] Not only could Azorubine and BB-FCF be moved across a separationmembrane from sample stream to the product stream, these two moleculescould also be separated from each other. With only 293 Da difference insize, the two compounds were separated from each other using a sizeexclusion separation where the largest molecule was retained in theproduct stream whilst the smaller molecule was allowed to transferthrough into the buffer stream. The following cartridge configurationwas used for the separation:

[0122] 5 kDa polyacrylamide restriction membrane

[0123] Support Grid

[0124] 3 kDa ultrafiltration separation membrane

[0125] 5 kDa polyacrylamide separation membrane

[0126] Support Grid

[0127] 1 kDa ultrafiltration restriction membrane

[0128] 5 kDa polyacrylamide restriction membrane

[0129] BB-FCF was separated from Azorubine. By allowing Azorubine topass through the 3 kDa ultrafiltration membrane whilst retaining theBB-FCF in the sample stream, an adequate separation was achieved. Theconcentration of Azorubine decreased significantly from the samplestream but did not build up substantially in the product stream. Thiswas due to loss into the buffer stream over time. The BB-FCF can passthrough a 3 kDa membrane but only very slowly so separation of the twomolecules was achieved. The movement of both molecules was monitoredusing 603 nm for BB-FCF and 516 nm for Azorubine. FIG. 9 shows theselective nature of the separation.

[0130] To improve the separation of BB-FCF from Azorubine a differentcartridge configuration was utilised as follows:

[0131] 5 kDa polyacrylamide restriction membrane

[0132] Support Grid

[0133] 200 kDa polyacrylamide separation membrane

[0134] Support Grid

[0135] 1 kDa ultrafiltration restriction membrane

[0136] 5 kDa polyacrylamide restriction membrane

[0137] This separation allowed both molecules to quickly transfer to theproduct stream from the sample stream. Then over time the Azorubinepassed into the buffer stream, whilst the BB-FCF was retained. The factthat the Azorubine would transfer through the 1 kDa Omegaultrafiltration membrane enabled separation of Azorubine from BB-FCFmore effectively than previously. FIG. 10 shows that after a three hourseparation close to 74% of the BB-FCF was still present in thedownstream whilst only 15% of the Azorubine remained. This separationshows the highly selective nature of the separation, which opens manypossibilities for its use with a number of different molecularseparations.

[0138] In new electrophoresis system it has been found thatmicromolecules under 5 kDa can be separated with this technology. Whenthe polyacrylamide membranes are used in combination with certaincommercially available membranes, molecules as small as ˜200 Da can beseparated and purified.

[0139] There were several problems encountered in the separation ofmicromolecules using an unmodified electrophoresis system. Difficultyretaining micromolecules in the system has been overcome with theaddition of combinations of membranes. However, these membranesthemselves posed problems in that they are not designed to retainliquids and can produce large levels of electro-endo-osmosis. The liquidretention problem has been solved by backing the membranes with thehydrogel polyacrylamide membranes, which also helped to reduce theelectro-endo-osmosis levels.

[0140] Several examples demonstrating the capability of the presentinvention in separating macromolecules have been shown though many otherpossible micromolecules of commercial interest do exist. For examplethis technology could be used in the separation and purification ofcytokines and growth factors for use in the pharmaceutical and researchindustries. Currently cytokines and growth factors account for over 50%of the biotechnology based pharmaceutical product sales. Other areaswhere the use of the present technology could improve current separationstrategies and be of substantial commercial benefit include purificationof pharmaceutical drugs, food additives, agro-chemicals and finechemicals.

[0141] It will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the invention as broadly described. The present embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

1. An electrophoretic separation apparatus for separatingmicromolecules, the apparatus comprising: (a) an anode; (b) a cathodedisposed relative to the anode so as to be adapted to generate anelectric field in an electric field area therebetween upon applicationof a voltage potential between the anode and the cathode; (c) aseparation membrane disposed in the electric field area; (d) a firstrestriction membrane disposed between the anode and the separationmembrane so as to define a first interstitial volume therebetween; (e) asecond restriction membrane disposed between the cathode and theseparation membrane so as to define a second interstitial volumetherebetween; and (f) means adapted to provide a sample constituent in aselected one of the first and second interstitial volumes; wherein uponapplication of the voltage potential, a selected separation product isremoved from the sample constituent, thorough the separation membrane,and provided to the other of the first and second interstitial volumesand wherein a micromolecule is capable of being retained in at least oneof the interstitial volumes.
 2. The apparatus according to claim 1wherein at least one restriction membrane is formed as a compositearrangement with at least two materials.
 3. The electrophoreticseparation apparatus of claim 1 wherein at least one of the restrictionmembranes is comprised of at least two membranes having distinctivepores sizes.
 4. An apparatus for electrophoretic separation ofmicromolecules, the apparatus comprising: (a) an anode buffercompartment and a cathode buffer compartment; (b) electrodes positionedin the buffer compartments; (c) a first chamber and a second chamberpositioned on either side of an ion-permeable separation membrane havinga defined molecular mass cut-off, the first chamber and the secondchamber being positioned between the anode and the cathode buffercompartments and separated by an ion-permeable restriction membranepositioned on at least one side of the separation membrane, therestriction membrane allowing flow of ions into and out of thecompartments and chambers under the influence of an electric field butsubstantially restrict movement of at least one micromolecule type fromthe second chamber into the buffer compartment.
 5. The apparatusaccording to claim 4 wherein the ion-permeable separation membrane has amolecular mass cut-off greater than the molecular mass of themicromolecule to be separated.
 6. The apparatus according to claim 4wherein at least one buffer compartment, sample chamber or productchamber is configured to allow flow of the respective buffer, sample orproduct solution to form a stream.
 7. The apparatus according to claim 4wherein at least one restriction membrane is formed as a compositearrangement with at least materials.
 8. The apparatus according to claim4 wherein at least one restriction barrier is formed as a sandwicharrangement with at least two layers of material.
 9. The apparatusaccording to claim 8 wherein the sandwich arrangement includes an innerlayer comprising a membrane having a pore size with a molecular masscut-off less than the about 5000 Da and an outer layer comprising amembrane having a molecular mass cut-off of greater than about 5000 Da.10. The apparatus according to claim 9 wherein the inner layer is madefrom an ultrafiltration, electrodialysis or haemodialysis membranematerial and the outer layer is a membrane material made frompolyacrylamide.
 11. The apparatus according to claim 10 wherein theultrafiltration membrane has a molecular mass cut-off between 100 Da and5000 Da.
 12. The apparatus according to claim 11 wherein theultrafiltration membrane has a molecular mass cut-off of about 1 kDa.13. The apparatus according to claim 4 wherein the ion-permeableseparation membrane is made from polyacrylamide and having a molecularmass cut-off from 5 to 1000 kDa.
 14. A separation cartridge suitable foruse in an electrophoretic separation apparatus for separatingmicromolecules, the cartridge comprising: (a) a housing; (b) anion-permeable separation membrane having a defined molecular masscut-off positioned in the housing; (c) an ion-permeable restrictionmembrane positioned either side of the separation membrane in thehousing and spaced to form a first chamber and second chamber on eitherside of the separation membrane, wherein the restriction membrane isadapted to allow flow of ions into and out of the compartments andchambers under the influence of an electric field but substantiallyrestrict movement of at least one micromolecule type from the secondchamber.
 15. The cartridge according to claim 14 further including: (d)electrodes positioned in the housing on the outer sides of therestriction barriers.
 16. The apparatus according to claim 14 whereinthe ion-permeable separation membrane has a molecular mass cut-offgreater than the molecular mass of a micromolecule to be separated. 17.The cartridge according to claim 14 wherein the separation membrane iscomposed of polyacrylamide and having a molecular mass cut-off fromabout 5 to 1000 kDa.
 18. The cartridge according to claim 14 wherein atleast one restriction membrane is formed as a composite arrangement withat least two materials.
 19. The cartridge according to claim 14 whereinat least one restriction membrane is formed as a sandwich arrangement ofmembranes with at least two layers of material.
 20. The cartridgeaccording to claim 19 wherein the sandwich arrangement includes an innerlayer comprising a membrane having a pore size with a molecular masscut-off less than the about 5000 Da and an outer layer comprising amembrane having a molecular mass cut-off of greater than about 5000 Da.21. The cartridge according to claim 19 wherein the inner layer is madefrom an ultrafiltration, electrodialysis or haemodialysis membranematerial and the outer layer is a membrane material made frompolyacrylamide.
 22. The cartridge according to claim 20 wherein theultrafiltration membrane has a molecular mass cut-off between 100 Da and5000 Da.
 23. The cartridge according to claim 22 wherein theultrafiltration membrane has a molecular mass cut-off of about 1 kDa.24. The cartridge according to claim 23 wherein the ion-permeableseparation barrier is a membrane made from polyacrylamide and having amolecular mass cut-off from 5 to 1000 kDa.
 25. A method of separating amicromolecule from a liquid sample, the method comprising: (a) providingan electrophoresis apparatus according to clam 4; (b) placing the samplein the first chamber of the apparatus; (c) selecting a solvent for thefirst chamber having a pH such that the micromolecule to be separated ischarged; (d) applying an electric potential between the first and secondchambers causing movement of micromolecules in the first stream throughthe separation membrane into the second chamber while unwanted moleculesare substantially prevented from entering the second chamber; (e)optionally, periodically stopping and reversing the electric potentialto cause movement of molecules having entered the separation membrane tomove back into the first chamber, while substantially not causing anymicromolecules that have entered the second chamber to re-enter firstchamber; and (f) maintaining steps (d) and optionally (e) until thedesired amount of micromolecules are moved to the second chamber. 26.The method according to claim 25 wherein the micromolecule is selectedfrom the group consisting of biotin, Brilliant Blue FCF (BB FCF),azorubine, phytoestrogen, digoxigenin, hormones, cytokines, dyes,vitamins, chemicals, neutraceuticals, pharmaceuticals food dietsupplements, and combinations thereof.
 27. The method according to claim25 wherein the sample is selected from the group consisting of crudeextracts, microbial cultures, cell lysates, cellular products, chemicalprocessing mixtures, cell culture media, plant products or extracts. 28.The method according to claim 25 wherein the solvent is Tris Boratebuffer around pH
 9. 29. The method according to claim 28 wherein bufferhas a concentration of 10 mM to 200 mM.
 30. The method according toclaim 29 wherein the buffer has a concentration of 20 mM to 80 mM.
 31. Amicromolecule purified or separated by the method according to claim 25.32. The micromolecule according to claim 31 selected from the groupconsisting of biotin, Brilliant Blue FCF (BB FCF), azorubine,phytoestrogen, digoxigenin, hormones, cytokines, dyes, vitamins,chemicals, neutraceuticals, pharmaceuticals, food supplements, andcombinations thereof.